Germinal center cytokine driven epigenetic control of Epstein-Barr virus latency gene expression

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.


Introduction
Epstein-Barr virus (EBV) persistently infects >95% of adults worldwide.Although typically benign, EBV nonetheless contributes to approximately 1.5% of all human cancers [1].These include endemic Burkitt lymphoma (BL), Hodgkin lymphoma, natural killer/T cell lymphoma, post-transplant lymphoproliferative disease (PTLD), primary central nervous system lymphoma and diffuse large B-cell lymphoma, which typically arise from the germinal center (GC) [1][2][3].EBV is also highly associated with multiple sclerosis [4,5].According to the EBV GC model, EBV uses distinct combinations of latent membrane proteins (LMP) and Epstein-Barr nuclear antigens (EBNA) to expand the pool of infected B-cells, navigate the B-cell compartment and promote infected cell differentiation into memory B-cells, the reservoir for lifelong infection [6].Across these latency programs, ~80 viral lytic antigens are largely silenced by epigenetic mechanisms.
The EBV genome is epigenetically programmed upon B cell infection [7][8][9][10].While EBV genomic DNA is epigenetically naïve in viral particles, it is rapidly chromatinized as incoming viral genomes reach the infected cell nucleus [7,11].Histone epigenetic marks, DNA methylation and three dimensional EBV genomic architecture then serve as major regulators of EBV gene expression.Much remains to be learned about host cell transcription factors and their upstream pathways in control of EBV epigenomic programming.The viral W promoter (Wp) drives an initial burst of EBNA expression, in particular EBNA2 and EBNA-LP, which highly upregulate MYC and other key B-cell targets [12][13][14][15][16][17][18][19].Infected cells then transition to the latency IIb program, in which the EBV genomic C promoter (Cp) drives expression of a transcript encoding EBNAs 1, 2, 3A, 3B, 3C and LP, whose messages are subsequently spliced.Shortly thereafter, EBNA2 activates the latent membrane promoters, driving expression also of LMP1 and LMP2A, culminating in the latency III program [3].If left unchecked, the transforming latency III program converts B-cells into immortalized lymphoblastoid cell lines (LCL), a key model for PTLD and AIDS-associated immunoblastic lymphomas [1,9,20].
Latency III drives cells into GC, where immune pressure together with incompletely understood mechanisms are believed to drive the transition to the EBV latency II program, comprised of EBNA1, LMP1 and 2A [9].EBNA1 expression is driven by the viral genome Q promoter (Qp) in latency II.Much remains to be understood about the precise GC signals and their downstream epigenetic mechanisms that culminate in Cp silencing, while instead supporting LMP expression in the absence of EBNA2 transcription activation.Upon memory Bcell differentiation, epigenetic mechanisms likely including DNA methylation and polycomb repressor complex 1 silence the LMP promoters to enable progression latency I program, where EBNA1 is the only EBV-encoded protein expressed [8,21].
The GC is a dynamic secondary lymphoid tissue microstructure, where T follicular helper (Tfh) and follicular dendritic cells (FDC) together with antigens drive B-cell responses [22,23].Tfh cytokines, including IL-2, 4, 10, and 21, together with the FDC derived cytokines IL-6, 15 and 27, are critical for GC establishment and maintenance, as well as for GC B-cell fate [23][24][25][26].Cytokines bind to plasma membrane B-cell receptors to activate Janus kinase (JAK) or Tyrosine kinase 2 (TYK2), which phosphorylate specific signal transducer and activator of transcription (STAT) family proteins.Phosphorylation drives STAT dimerization via reciprocal SH2 domain-phosphotyrosine interactions and nuclear translocation to enable target gene regulation (S1A Fig) [27][28][29].IL-21 decreases EBNA2 expression in latency III B cells [30,31], suggesting a potential GC cytokine role in driving the transition from latency III to II.Moreover, IL-4, 10, and 21 each de-repress LMP1 expression in newly infected cells and in latency I Burkitt and natural killer (NK) lymphoma cells [30][31][32][33][34][35][36], further suggesting roles in support of latency II.IL-15 also drives NK and T-cell responses against EBV transformed peripheral blood B-cells [37,38], potentially suggesting that it may enhance immune pressure against latency III B-cells within the GC.However, much remains to be learned about the mechanisms by which cytokines secreted by Tfh and FDC alter the EBV epigenome to repress EBNA but instead support LMP expression.
To gain insights into mechanisms by which GC cytokines alter EBV latency gene expression and the viral epigenome, we systematically screened effects of Tfh and FDC cytokines on EBV latency gene expression.Tfh cytokines, including IL-4, 10 and 21, each upregulated LMP1 but downregulated EBNA2 and 3 levels in B cells with latency III.By contrast, the key FDC cytokine IL-15 diminished Cp driven EBNA expression but did not significantly alter LMP1 levels.CRISPR analysis identified that STAT3 and to a lesser extent STAT5 were critical for these cytokine effects on EBNA and LMP1 expression.Taken together, our results highlight GC cytokines driven STAT3 and 5 remodeling of the EBV epigenome to support the latency III to latency II program transition.

GC cytokines support the latency II transition
To systematically characterize GC cytokine effects on EBV latency gene expression, we incubated the LCL GM12878 with a panel of Tfh-derived cytokines, IL-2, IL-4, IL-10 or IL-21.In parallel, we incubated GM12878 with the FDC-derived cytokines IL-6, IL-15 or IL-27 for 0, 2, 4 or 6 days (Figs 1A and S1A).While it is not known how long EBV+ B-cells reside within the GC, it is likely that they remain present for at least several days, in order to proliferate and differentiate into memory B cells, and GC structures themselves persist for weeks to months.Cytokine effects on EBV latency programs were defined by immunoblot for EBNA2, EBNA3C and LMP1, since this panel of EBV oncoproteins can be used to assign the latency program.Interestingly, most of these cytokines reduced EBNA2 and 3C expression, though results were the most pronounced for IL-21, which rapidly and robustly impaired EBNA2/3C expression (Fig 1B  We next performed RNA-seq analysis to systematically characterize IL-15 and IL-21 effects on EBV genome-wide expression, as representative of FDC vs Tfh cytokine signaling, respectively.After six days of treatment, IL-15 significantly decreased expression of multiple latency III genes, including EBNA2, EBNA3, EBNA-LP, LMP1 and LMP2s, but increased expression of a subset of lytic cycle genes, including immediate early BZLF1 and early BMRF1 (Fig 1C  ).
GC cytokines alter B-cell gene expression patterns via multiple effectors, including distinct JAK/STAT pathways.As expected, the panel of cytokines differentially activated STATs, including STAT5 activation by IL-2 and IL-15 versus STAT6 activation by IL-4 versus STAT3 activation by IL-6, IL-10, IL-21 and IL-27, as judged by immunoblot, using well characterized phosphorylation marks of STAT activation [26,27] (Figs 1E and S1B-S1C).Consistent with our RNA-seq analyses, IL-15 de-repressed BZLF1 and BMRF1 expression at the protein level, as did IL-2 (Fig 1E), suggesting that it induces an abortive lytic cycle in at least a subset of cells.Notably, these two cytokines share receptor beta and gamma chain subunits, which are transmembrane proteins that activate downstream pathways, including JAK/STAT [41].
EBV-infected B-cells likely also experience combinatorial cytokine signaling in germinal center microenvironments.To model these effects, we next treated GM12878 and GM12881 We next asked the extent to which Tfh and FDC cues can alter EBV latency gene expression within the latency I B-cell context.While several Tfh signals, including IL-4+CD40L, IL-10 or IL-21 can each de-repress LMP1 expression in B-cells with the latency I program [30][31][32][33][34], it has remained unknown the extent to which other GC microenvironmental cues more broadly alter EBV latency gene expression within latency I. To gain insights, we treated latency I Mutu I and Kem I Burkitt cells with a panel of Tfh and FDC cytokines, as there is no primary human B-cell latency I models currently available.IL-21 strongly activated STAT3, as judged by tyrosine 705 phosphorylation, and robustly de-repressed LMP1 expression in Mutu I and Kem I (Figs 1F and S2C and S2D).By contrast, IL-4+CD40L or IL-10 treatment also induced STAT3 phosphorylation and LMP1 expression, albeit to a lesser extent (Fig 1F).This did not appear to be a full transition to the latency II program, as neither IL-10 nor IL-21 induced LMP2A to an appreciable degree in Mutu I or Kem I (S2E Fig) .Differences between GC cytokine STAT activation in the latency I vs III context may relate to altered expression of receptors versus negative regulators of JAK/STAT signaling.
To then systematically analyze latency I B-cell responses to the Tfh signals IL-4+CD40L vs IL-21, we performed RNA-seq on Mutu I that were mock-stimulated or stimulated by these Tfh cues for 1 day.This early timepoint was chosen since we observed robust effects on LMP1 de-repression by that early timepoint, and as we observed reduced Mutu I viability with longer treatments.Consistent with our immunoblot analysis, IL-4+CD40L only modestly increased LMP1 expression, whereas IL-21 strongly induced LMP1 (Fig 1G and 1H).Notably, these stimuli did not significantly de-repress expression of EBNA or mildly increased LMP2 mRNAs, suggesting a specific effect at the level of the LMP1 promoter.
Analysis of Mutu I host transcriptome responses to either IL-4+CD40L or IL-21 treatment highlighted upregulation of multiple LMP1 target genes [42], including mRNAs encoding the NF-κB subunits RelB and p100/52 (encoded by NFKB2), ICAM-1 and IRF4 (S2F and S2G Fig) .The NF-κB pathway signaling pathway and EBV infection were amongst the pathways most highly enriched by either cytokine treatment.While direct effects of the cytokines themselves may account for a subset of these changes, we note that IL-21 is not a strong inducer of NF-κB signaling, suggesting that de-repressed LMP1 may be an important mediator of the observed host transcriptomic changes.

STAT3 and STAT5 mediate GC cytokine effects on the EBV latency III program
We next investigated effects of chemical or CRISPR JAK/STAT blockade to gain further insights into specific STAT roles in control of EBV latency gene expression downstream of IL-15 and IL-21.First, to broadly characterize JAK/STAT roles in LMP and EBNA expression, we treated latency III GM12878 and Jijoye cells with IL-15 or IL-21, in the absence or presence of the pan-JAK ATP-competitive inhibitor CAS 457081-03-7 (also referred to as JAK inhibitor I or JAKi).On-target JAKi effects were confirmed by immunoblot analysis of Taken together, these results suggest that STAT3 and 5 have partially redundant roles in cytokine mediated EBNA2/3C repression, perhaps through the action of STAT3/5 heterodimers, whereas STAT3 is a major driver of IL-21 driven LMP1 hyper-induction, with relevance to latency III to II reprogramming in the GC microenvironment.

STAT and DNA methylation role in latency I LMP1 de-repression by GC cytokines
To gain insights into JAK/STAT roles in GC cytokine triggered LMP1 de-repression in latency I B-cells, we treated Mutu I or Kem I Burkitt cells with IL-10, IL-21 or IL-4 together with CD40 ligand, in the absence or presence of JAK inhibition.We tested these GC stimuli since each hyper-induced LMP1 and robustly induced STAT phosphorylation in latency I cells and had previously been reported to de-repress LMP1 expression from latency I [30][31][32][33][34][35][36] (Fig 1F).JAKi treatment strongly impaired LMP1 upregulation by each of these stimuli (Figs 3A and S5A), consistent with a key JAK/STAT role in epigenetic regulation at the level of the LMP1 promoter.
To next gain mechanistic insights into specific STAT roles, we tested effects of CRISPR depletion of STAT transcription factors that were highly phosphorylated in response to these GC stimuli (Fig 1F

GC cytokines remodel epigenetic status of C promoter
Multiple GC cytokines repressed latency III EBNA expression, suggestive of epigenetic effects at the level of Cp, which drives the large EBV transcript encoding all six EBNAs.
We performed ChIP to characterize how IL-15 and IL-21 alter STAT3 versus STAT5 occupancy at two predicted STAT binding sites using PROMO online tool [59,60], located at 300

JAK/STAT signaling roles in newly infected B-cell latency gene expression and transformation
JAK/STAT signaling contributes to EBV latency gene expression in newly infected primary human B-cells [61,62].Notably, STAT3 tyrosine 705 phosphorylation is elevated within 30 minutes of primary B-cell exposure to EBV, and total STAT3 protein levels are upregulated within 8 hours of EBV infection [62,63].Such early effects on STAT3 are likely triggered by EBV interaction with B-cell receptors and/or internalization and preceded EBV latency gene expression [62,63].To build upon these data, we characterized STAT3 and STAT5 phosphorylation levels over multiple subsequent timepoints, in which EBV converts primary human cells into lymphoblastoid cells.Purified CD19+ human peripheral blood B cells were isolated by negative selection and infected with EBV.Timecourse analysis was then performed to measure levels of EBV latency gene, STAT3 and STAT5 expression, as well as of STAT3 and 5 phosphorylation, to indicate their activation status.Interestingly, EBV upregulated STAT3 and STAT5A levels, in particular between days 4 and 21 post-infection, whereas STAT5B levels were relatively constant.Whereas EBV triggered STAT3 phosphorylation, in particular between days 4 and 21 post-infection, a period in which LMP1 levels were markedly elevated and EBNA2 and 3C levels diminished (Fig 6A).Notably, EBV did not trigger STAT5 phosphorylation, as judged by immunoblot of phosphotyrosine 694 (Fig 6A).
We next investigated the effects of IL-21 on EBV latency gene expression when dosed at day 7 post-infection, the earliest timepoint when B-cells begin to convert to lymphoblastoid physiology [9,64] To then gain insights into germinal center cytokine effects on EBV-infected primary Bcells, we treated cells at 7 days post-infection with vehicle control, IL-4, IL-15, IL-21, IL-27 or multiple combinations thereof.IL-15 or IL-21 were each sufficient to reduce EBNA2 and EBNA3C expression relative to levels in untreated cells, but we did not appreciate additive effects of combinatorial cytokine treatment on their expression (Fig 6B).While IL-4 did not appreciably alter EBNA2 or EBNA3C levels, it was able to boost LMP1 protein expression above control cell levels.Interestingly, the combination of Tfh cytokines IL-4 and IL-21 hyperinduced LMP1 expression, whereas addition of the FDC cytokine IL-15 to IL-21 did not

PLOS PATHOGENS
appreciably alter LMP1 levels relative to those observed with IL-21 treatment alone.Combinatorial treatment with the FDC cytokines IL-15 and IL-27 did not boost LMP1 levels relative to untreated cells (Fig 6B).Immunoblot analysis demonstrated the expected phosphorylation of STAT1, 3, 5 and 6 in response to each of these cytokines, suggesting that their pathways were similarly activated by cytokines in the primary cell as well as transformed B cell settings (Fig 6B ).
To characterize the roles of JAK/STAT signaling in EBV-mediated B-cell transformation, we treated newly infected primary human B-cells with JAKi at 4, 7 or 10 DPI.Consistent with JAK/STAT downmodulation of EBNA2 and EBNA3 expression at these early times post-infection, JAKi treatment increased EBNA2 and EBNA3C expression (Fig 6C).Surprisingly, JAKi treatment also mildly increased LMP1 expression, which likely occurred secondary to increases in EBNA2 levels.JAKi treatment also impaired outgrowth of EBV-infected B-cells in a transformation assay (Fig 6D ), suggesting that EBV-driven JAK/STAT signaling supports Bcell immortalization, potentially by titrating the levels of EBV oncoprotein expression.Taken together, our results support a model in which JAK/STAT signaling exerts control over EBV latency gene expression through epigenetic effects on key EBV latency gene promoters (Fig 7).

Discussion
The EBV germinal center model posits that microenvironmental cues trigger latency program remodeling in order to support infected B-cell survival, immunoevasion and memory B-cell differentiation [6].Yet, knowledge has remained incomplete about how specific Tfh and FDC signals alter EBV latency gene promoter epigenomes.Here, we present the first CRISPR analyses to dissect specific STAT roles in latently EBV-infected B-cell responses to GC cytokine Cytokines secreted by germinal center FDC and Tfh cells trigger JAK/STAT signaling to drive STAT homodimer or heterodimer nuclear translocation.STAT dimers bind to the C promoter together with a co-repressor to increase levels of repressive 5-methylcytosine (5-mC), H3K9me2/3 and H2AK119Ub epigenetic marks, while decreasing activating H3K27Ac marks.C promoter inactivation decreases latency III EBNA expression.By contrast, STAT dimers bind to the LMP1 promoter together with a co-activator to drive epigenetic remodeling to increase levels of LMP1 promoter H3K27Ac and to decrease levels of H2AK119Ub, which supports LMP1 expression despite concurrently increased 5mC levels.By contrast, GC cytokines increase the abundances of LMP2 promoter repressive H2AK119Ub, H3K9me3 and 5mC marks to downmodulate LMP2 expression from levels observed in latency III.(BioRender was used to create the schematic models).https://doi.org/10.1371/journal.ppat.1011939.g007cues.We highlight crosstalk between EBV genomic STAT occupancy, histone modification and DNA methylation in GC-cytokine driven reprograming.Our data support a model in which GC cytokines drive a STAT3/5 dependent transcription repressive complex at the EBV genomic C promoter, but instead drive a STAT3 dependent transcription activation complex at the LMP1 promoter (Fig 7).STAT3/5 heterodimers may serve to nucleate a transcription repressive complex at Cp, whereas STAT3 homodimers may instead promote transcription activation at LMP1p.Since STAT3 homodimers support EBNA1 expression in latency II [58,67], STAT signaling provides a key means by which EBV-infected cells translate GC microenvironmental cues to the epigenome.
We recently identified that DNA methylation is sufficient for silencing Cp-driven EBNA expression, but that DNA methylation and PRC1 are each important for silencing LMP1 and LMP2A expression in latency I Burkitt cells [8,21].It is therefore noteworthy that IL-21 increased C, LMP1 and LMP2 promoter DNA methylation, but decreased the PRC1 H2AK119Ub mark only at the LMP1 promoter in a STAT3/5 dependent manner.IL-21 may alter LMP1 promoter H2AK119Ub abundance by promoting dismissal of PRC1 from LMP1p or instead by recruiting the H2AK119Ub erasers BAP1 or USP16 [68] in a STAT3/5 dependent manner.Importantly, EBNA2 induces and also recruits the TET2 demethylase to the C and LMP promoters [69,70].Therefore, EBNA2 downmodulation by IL-21 likely contributed to the observed increase in EBV genomic methylation.
How then is LMP2A supported in the GC microenvironment upon GC cytokine driven EBNA2 repression?While we studied responses to individual cytokine cues, it is possible that combinatorial signals may be needed to support LMP2A expression.Alternatively, a distinct GC microenvironmental cue not modelled in our study may be required to support LMP2A expression, such as from dendritic or regulatory T cells.Thus, a prediction of this model is that on a single cell level, subsets of EBV-infected cells may express LMP1, LMP1 together with LMP2A, or perhaps only LMP2A within distinct GC microenvironmental niches.Such flexibility may support evasion from cytotoxic T-cell responses directed at either LMP1 or LMP2A, may alter the extent of infected cell proliferation or residence time within the GC, and/or may support GC exit upon memory B-cell differentiation into the EBV memory cell reservoir.
Latent EBV infection supports B-cell JAK/STAT signaling, which may provide a basal level to calibrate latency gene expression, even in the absence of Tfh or FDC derived cytokines.The LMP1 C-terminal activation region 3 binds to JAK3 [71], though this region of LMP1 may not by itself be sufficient to activate JAK/STAT signaling [72,73].LMP1 induces IL-10 expression in vitro [74] and together with LMP2A in germinal center B-cells in vivo [75], though levels are likely to be lower than those secreted by Tfh in the GC microenvironment.Furthermore, EBV driven reactive oxygen species accumulation further supports STAT3 activation in the early stages of EBV-driven B-cell outgrowth [61].Thus, EBV may have evolved to require a high threshold of JAK/STAT signaling to ensure that latency program selection occurs in the GC microenvironment on the pathway to memory cell differentiation.
While our data suggest that STAT3 is critical for GC cytokine induced remodeling towards latency II, it is noteworthy that IL-21 more strongly induced LMP1 than IL-6, IL-10 or IL-27, which also strongly activated STAT3.One model to reconcile these observations is that IL-21 signaling may induce a higher abundance of STAT3 homodimers within latency III cells, and these are required for the observed effects on LMP1 expression.Alternatively, IL-21 may more strongly induce co-activators that together with STAT3 upregulate LMP1 expression.
Gamma-herpesviruses may have evolved to subvert STAT3 signaling to support GC-dependent differentiation.EBV, Kaposi's Sarcoma Associated Herpesvirus and murine gammaherpesvirus 68 (MHV68) have each evolved mechanisms to activate STAT3 [62,79,[87][88][89][90][91].STAT3 is important for the establishment of longterm latency by MHV68 [92].However, in contrast to our findings for EBV, STAT3 does not directly regulate MHV68 viral gene expression, but instead dampens type I IFN responses in newly infected B-cells [93].Thus, EBV has evolved specific mechanisms to coopt B-cell STAT signaling to modulate latency gene expression in response to B-cell cues.It is not presently known whether EBV+ B-cells enter GC dark zone structures, in which B-cells undergo multiple rounds of proliferation and somatic hypermutation following stimulation by Tfh and FDC within light zone regions.Since GC cytokine stimulation and STAT3 phosphorylation take place within light zones [94], it is plausible that EBV + B-cells may express higher LMP1 levels within light zones, and that they may therefore predominantly reside within GC light zone regions.However, single cell analyses of EBV-infected secondary lymphoid tissue have not yet been performed to address this open area.
Many EBV-associated cancers are currently treated with high intensity chemotherapy or radiation therapy, which causes substantial morbidity and increases the risk of secondary malignancies.Likewise, use of anti-CD20 B-cell depleting antibodies such as rituximab to target EBV-driven lymphoproliferative diseases can be complicated by lymphopenia, variable Bcell recovery, hypogammaglobulinemia, prolonged neutropenia and rare but fatal viral reactivation [95,96].Therefore, novel therapeutic approaches are needed to more selectively target a range of EBV-associated cancers.In depth understanding of the molecular mechanisms that control EBV latency gene expression may lay the foundation for rational therapeutic approaches that target specific EBV-associated cancers, either by depriving tumor cells of necessary EBV oncogene expression in the case of lymphomas with the latency III program or by de-repressing immunogenic latency protein antigens in tumors with more highly restricted forms of latency, such as Burkitt lymphoma.
With regards to tumors dependent on latency III such as post-transplant lymphoproliferative disease or central nervous system lymphoma, it may be feasible to target JAK/STAT signaling to downmodulate EBNA expression.An intriguing possibility is that targeting the JAK/ STAT pathway with clinically approved small-molecule inhibitors such as ruxolitinib, currently used treatment of graft-versus-host disease [97] and for other hematological disorders, could be tested as a prophylactic or preemptive approach for the prevention of EBV-driven PTLD.While EBV-driven lymphomas can emerge in patients treated with potent T-cell immunosuppression together with ruxolitinib, it might nonetheless confer protection against the outgrowth of EBV-driven polymorphic B-cell clones.
Conversely, cytokine therapy or epigenetic approaches could be developed to derepress LMP1 in order to sensitize latency I tumors such as Burkitt lymphoma to antiviral T-cell surveillance, including adoptive transfer of T-cells reactive with LMP1 derived epitopes [98,99].Not only is LMP1 itself a targetable tumor antigen, but once de-repressed, it also stimulates potent T-cell responses to tumor associated antigens [100].In addition, IL-15 and IL-21 enhance natural killer and CD8+ T cell cytotoxicity and antitumor effects [101][102][103].An interesting approach could therefore be to combine HLA-matched, third party cytotoxic CD8+ Tcells [104] with IL-21, perhaps as an Fc-fusion to enhance [105].Indeed, IL-21 therapy appears to generally be well tolerated [106].In this manner, IL-21 treatment could not only induce LMP1 expression in Burkitt tumors, but could also enhance cellular immune responses to EBV-infected tumor cells.
Bispecific antibodies that target the T-cell receptor (TCR) and an HLA-presented LMP2A peptide is a promising approach which has demonstrated activity against latency III EBV+ Bcell murine xenografts [107].It may therefore be possible to combine IL-21 with a bispecific antibody targeting the TCR and LMP1 derived peptides to target EBV+ Burkitt tumors.Further studies are required to explore whether IL-21 or other germinal center cytokines might likewise de-repress or boost LMP1 expression in EBV+ epithelial cell tumors, including gastric and nasopharyngeal carcinoma.
IL-27 receptor signaling was recently found to play a key role in the stimulation of CD8+ Tcell anti-EBV responses, as congenital biallelic loss-of-function variants in the IL-27 receptor alpha chain are associated with severe primary EBV infection.Interestingly however, EBVtransformed B-cells secrete IL-27, and an autocrine loop between IL-27 receptor+ T-cells and IL-27 secretion by infected B-cells is important for EBV-transformed B-cell proliferation in vitro [108].Thus, the IL-27 receptor/IL-27 axis could potentially constitute another therapeutic target.Further studies will be required to determine whether there may be a role for IL-27 therapy, for instance to boost anti-EBV T-cell responses.While IL-27 supports EBV-transformed B-cell proliferation at doses secreted by infected cells in culture, we found that at high doses, IL-27 instead downmodulates EBNA expression, which might also provide anti-tumor effects by impairing transformed cell growth in vivo.
In summary, multiple FDC and Tfh derived cytokines repress Cp driven EBNA expression, whereas IL-21 and to a lesser extent IL-4 and IL-10 support LMP1 expression through STAT dependent EBV epigenomic remodeling.STAT3 and 5 were critical for cytokine mediated Cp silencing, whereas STAT3 was critical for LMP1 hyper-induction.GC cytokine signaling increased repressive epigenetic marks, including DNA methylation and H2AK119Ub, while decreased active chromatin mark H3K27Ac at Cp in EBV latency III cells.However, IL-21 increased H3K27Ac at the LMP promoters, but decreased H2AK119Ub only at the LMP1 promoter in both EBV latency III and I cells.IL-21 also decreased DNA methylation at LMP1 promoter in EBV latency I Burkitt cells.Therefore, STAT3 and 5 serve as major hubs of EBV epigenomic remodeling in response to GC cytokine signaling to support latency program remodeling.

Materials and methods
Coriell Institute), Kem III (A gift from Dr. Jeffrey Sample) and Jijoye (purchased from American Type Culture Collection, ATCC), were all grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS).293T cells (purchased from ATCC) were grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS.All B cell lines used in this study stably express Streptococcus pyogenes Cas9, which were generated by lentiviral transduction followed by blasticidin selection [109].All cells were grown in a humidified chamber with 5% carbon dioxide at 37˚C.

Cytokines and JAKi treatment
Latency I and III B cells were seeded at 500,000 cells/ml in 12-well plates and mock treated with PBS or cytokines (S3 Table) at 50 ng/ml and 100 ng/ml, respectively.EBV infected human primary B cells were treated with IL-15 or IL-21 at 100 ng/ml.For long-term treatment, cells were re-seeded with fresh culture medium supplemented with indicated cytokines, which were refreshed every 48 hours.For JAK inhibitor treatment, cells were pre-treated with JAK inhibitor I (JAKi) at indicated doses for one hour at 37 ˚C, followed by cytokine treatment, refreshed every 48 hours.

cDNA cloning and transduction
cDNA entry vectors used in this study are listed in S4 Table, which were purchased from DNASU and Addgene.STAT1, 3, and 6 cDNA were sub-cloned into the destination vector pLX-TRC313 (a gift from John Doench) and STAT3_p.A662C_N664C (constitutively active STAT3 with A662C and N664C mutations) [44] was cloned into pLIX-402 (a gift from John Doench) by Gateway LR recombination.As described previously [113], the destination vector and donor vector containing the gene of interest were co-incubated with 1x LR Clonase Enzyme Mix overnight at room temperature.The reaction mixture was then transformed into Stbl3 competent cells and plated on LB agar plate containing ampicillin.Destination vectors were used to make lentiviruses, which were used to transduce target B-cells.Transduced cells

Immunoblotting
Immunoblotting analysis was performed as previously described [21], Cells were lysed in 1x Laemmli Sample Buffer and sonicated briefly.For detection of LMP2A, cells were lysed with M-PER Mammalian Protein Extraction Reagent and incubated on ice for 30 minutes.Lysates were centrifuged at 15,000 x g for 15 minutes.2x Laemmli Sample Buffer was added into supernatant and boiled at 70 ˚C for 10 minutes.Lysates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes, which were blocked with 5% nonfat milk in TBST buffer for 1 hour and then incubated with primary antibodies at 4 ˚C overnight.Blots were then washed 3 times with TBST, followed by secondary antibody incubation for 1 hour at room temperature.Blots were washed 3 times in TBST buffer and were developed with the ECL chemiluminescence substrate.Images were captured by a LI-COR Fc platform.All antibodies used in this study are listed in S3 Table.

Flow cytometry assay
Cells were washed once with FACS buffer (2% FBS v/v, PBS), followed by incubation with primary antibodies in FACS buffer for 30 minutes at room temperature in the dark.Labeled cells were palleted, washed twice and resuspended in FACS buffer into flow cytometry-compatible tubes and processed immediately.Flow cytometry data was recorded with a BD FACSCalibur instrument and analyzed with FlowJo X software.

Akata virus production, primary B cells isolation and infection
EBV was produced from EBV+ Akata cells.In brief, EBV+ Akata cells were resuspended in FBS-free RPMI media at 2-3 million cells/mL and induced with 0.25% (v/v) goat anti-human immunoglobulin G serum for 6 hours at 37 ˚C.Cells were then pelleted and resuspended in 4% FBS RPMI media and cultured in 37 ˚C for 3 days.Supernatant were then collected and filtered through 0.45 μM filter.Viruses were 50-fold concentrated by ultracentrifugation and stored at -80 ˚C until use.
Primary B-cells were isolated by negative selection from discarded, de-identified peripheral blood mononuclear cells from the Brigham and Women's Hospital Blood Bank, obtained following platelet donation, using an Institutional Review Board approved protocol and written consent was obtained from the donors.RosetteSep and EasySep negative isolation kits were used according to the manufacturer's instructions to isolate CD19+ B-cells.B cells were then cultured with RPMI containing 10% FBS.Primary B cells were seeded at 500,000 cells/ml and infected by the Akata EBV strain at multiplicity of infection (MOI) of 0.1, as determined by the Green Daudi assay.

Primary human B cell EBV transformation assay
EBV transformation assays were performed as described previously [114].Briefly, purified human primary B cells were infected with Akata EBV using serial 10-fold dilutions.Cells were cultured with media containing DMSO or JAKi (200 ng/ml) and were seeded in 96-wells plates at 500,000 cells/ml (30 wells per condition).Media containing DMSO or JAKi was refreshed every three to four days.The percentage of wells positive for B-cell outgrowth at four weeks post infection was calculated and plotted relative to the dilution of virus.

Chromatin immunoprecipitation (ChIP) assay
After cytokine treatment, 10 million cells were cross-linked with 1% formaldehyde in 10 ml growth medium for 10 minutes, followed by quenching with 2.5M glycine in distilled water for 5 minutes.Cells were washed with ice-cold PBS three times and then lysed in 0.5 ml 1% SDS lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS), supplemented with 1x cOmplete, EDTAfree Protease Inhibitor Cocktail.Chromatin was fragmented using a Bioruptor Pico sonication device with 30s on/ 30s off (20 cycles for GM12878 cells, 12 cycles for Mutu I cells), and centrifuged at 13,200 rpm for 10 mins at 4 ˚C.This protocol resulted in fragments of average length 100-200 bp, to enable differentiation of STAT occupancy at closely spaced EBV genomic STAT binding sites.Supernatants were removed and then diluted 1:10 in ChIP dilution buffer (1.2 mM EDTA, 16.7 mM Tris, 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100) supplemented with protease inhibitor cocktail.Chromatin from one million cells was used for each ChIP reaction.1% of sonicated chromatin was saved as input and stored at -80 ˚C until use.Diluted chromatin was rotated overnight at 4 ˚C with the indicated antibody and 20 μl protein A+G magnetic beads.Next day, beads were pelleted, washed twice with a lower salt buffer (150 mM NaCl, 2 mM EDTA, 20 mM Tris, 0.1% SDS, 1% Triton X-100) and then a high-salt buffer (500 mM NaCl, 2 mM EDTA, 20 mM Tris, 0.1% SDS, 1% Triton X-100), and once with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris) and finally TE buffer (10 mM Tris, 1 mM EDTA).Chromatin was eluted in Elution buffer (100 mM NaHCO3, 1% SDS) and reverse cross-linked at 65 ˚C for 2 hours.QIAquick PCR purification kits were used to purify the immunoprecipitated DNA, followed by qPCR with PowerUp SYBR green PCR master mix on a CFX Connect Real-Time PCR Detection System (Bio-Rad).All reagents, antibodies and primers used for ChIP are listed in S3 and S4 Tables.

Methylated DNA immunoprecipitation (MeDIP) assay
Genomic DNA was extracted using DNeasy Blood& Tissue Kit, followed with MeDIP assay with MagMeDIP kit, following the manufacturer's protocol.qPCR was then performed with primers specifically target EBV promoters.All reagents and primers used for MeDIP are listed in S3 and S4 Tables.

RNA-seq and data analysis
mRNA was isolated via the RNeasy Mini kit with in-column genomic DNA digestion protocol was followed, according to the manufacturer's instructions.To construct indexed libraries, 1 μg of total RNA was used for polyA mRNA purification, using the NEBNext Poly(A) mRNA Magnetic Isolation Module, followed by library preparation using the NEBNext Ultra RNA Library Prep with Sample Purification Beads.Each experimental treatment was performed in biological triplicate.Libraries were multi-indexed, pooled and sequenced on an Illumina NovaSeq 6000 using PE150 Sequencing Strategy by Novogene Corporation.Adaptor-trimmed reads were mapped to Akata EBV genome (Accession#: KC207813.1)or human GRCh37.83transcriptome assembly using salmon (v1.10.0).Quality control was performed using fastqc.Differentially expressed genes were identified in R (v4.0.3) using DESeq2 [115] under default settings with the apeglm shrinkage estimator (https://doi.org/10.1093/bioinformatics/bty895)and annotations derived from the hg19 build from Ensembl release 75 [116] and accessed via biomaRt.Volcano plots were generated in GraphPad Prism 8, using Log 2 (Fold Change) and −Log 10 (p value) data.Differentially expressed genes from each condition were subjected to Enrichr analysis and top 10 KEGG pathways with adjusted p value < 0.05 cutoff were visualized.All RNA-seq datasets were deposited to the NIH GEO omnibus with accession number GSE252480.All reagents and kits used for RNA-seq are listed in S3 ).By contrast, IL-10 and IL-21 upregulated LMP1 expression within 2 days of treatment (Fig 1B).Similar effects were observed in a second LCL, GM12881 (S1B Fig).IL-21 also

Fig 1 .
Fig 1. GC cytokines support the transition to EBV latency II.(A) GC schematic, illustrating key T follicular helper cell (Tfh) and follicular dendritic cell (FDC) secreted cytokines and CD40 ligand (CD40L) that signal to GC B-cells.(BioRender was used to create the schematic models.)(B) Immunoblot analysis of whole cell lysates (WCL) from GM12878 treated with the indicated cytokines for two, four, or six days.(C) Volcano plots of EBV gene expression from n = 3 replicates of GM12878 stimulated by IL-15 vs. mock-stimulated for 6 days.(D) Volcano plots of EBV gene expression from n = 3 replicates of GM12878 stimulated by IL-21 vs. mock-stimulated for 6 days.(E) Immunoblot analysis of WCL from GM12878 cells treated with the indicated cytokines for six days.(F) Immunoblot analysis of WCL from Mutu I cells treated with the indicated cytokine for one day or from GM12878 for comparison.(G) Volcano plot of EBV gene expression from n = 3 replicates of Mutu I stimulated by IL-4+CD40L vs. mock-stimulated for one day.(H) Volcano plot of EBV gene expression from n = 3 replicates of Mutu I stimulated by IL-21 vs. mockstimulated for one day.All cytokines were used at 100 ng/ml and 50 ng/ml in GM12878 vs Mutu I, respectively, and were refreshed every two days.Immunoblots are representative of n = 3 replicates.https://doi.org/10.1371/journal.ppat.1011939.g001 LCLs with the Tfh cytokines IL-4+IL-21, the FDC cytokine IL-15 together with IL-21 or the FDC cytokines IL-15 and IL-27 (Fig 1A).IL-15/IL-21 and to a lesser extent IL-15/IL-27 suppressed EBNA2 and EBNA3C expression somewhat more strongly than either cytokine alone.Interestingly, while IL-4/IL-21 did not repress either EBNA2 or EBNA3C more than IL-21 alone, IL-4/IL-21 did boost LMP1 levels beyond those observed with IL-21 or IL-4 alone (S2A and S2B Fig), likely due to alterations in JAK/STAT signaling.

Fig 2 .
Fig 2. STAT3/5 roles in EBV latency gene regulation by IL-15 or IL-21.(A-B) Immunoblot analysis of WCL from GM12878 (A) or Jijoye (B) cells pre-treated with DMSO vehicle or JAK inhibitor CAS 457081-03-7 (JAKi, 200 ng/ml) for one hour, followed by treatment with IL-15 or IL-21 for six days.(C-D) Immunoblot analysis of WCL from GM12878 cells expressing control sgRNA versus sgRNA targeting the indicated STAT3 and/or STAT5 genes, which were then mock treated or treated with IL-15 (C) or IL-21 (D) for six days.(E-F) Immunoblot analysis of WCL from GM12878 (E) or Jijoye (F) cells expressing cDNA encoding GFP or constitutive activated STAT3 (STAT-CA), whose expression was induced by 0.5 or 1 μg/ml doxycycline (Dox).(G) Methylated DNA immunoprecipitation and quantitative PCR (MeDIP-qPCR) analysis of GM12878 cells with control or STAT3/5A/5B sgRNA expression, which were mock treated or treated with IL-15 or with IL-21 for six days.Shown is mean ± standard deviation (SD) from n = 3 replicates of Cp qPCR signal.*p < 0.05; NS: not significant.IL-15 and IL-21 were used at 100 ng/ml throughout.Immunoblots are representative of n = 3 replicates.https://doi.org/10.1371/journal.ppat.1011939.g002 ). Depletion of either STAT1 or STAT3 blunted LMP1 de-repression by IL-4+CD40L stimulation or even by IL-4 alone(Figs 3B and S5B).By contrast, depletion of STAT3, but not STAT1, impaired IL-10 and IL-21 mediated LMP1 de-repression in Mutu I and Kem I cells (Figs3C and S5B).Thus, STAT1/3 heterodimers may be important for IL-4 driven LMP1 de-repression, whereas distinct STAT3 heterodimers or homodimers may mediate LMP1 de-repression downstream of IL-10 and IL-21.Consistent with the latter hypothesis, induction of the constitutively active STAT3 allele was sufficient to de-repress LMP1 expression in Mutu I (S5C Fig).Likewise, STAT3 and to a somewhat lesser extent STAT6 overexpression enhanced LMP1 de-repression in response to cytokine treatment (S5D Fig).To gain further insights into cytokine cross-talk with EBV-genomic CpG methylation, we next analyzed IL-21 effects on the abundance of DNA methyltransferase machinery.IL-21 downregulated expression of the de novo CpG methylation writer DNMT3B (Fig 3D), whose expression counteracts latency III gene expression [21].Likewise, IL-21 downmodulated UHRF1 expression (Fig 3D), which is important for maintenance of EBV genomic methylation marks, together with DNMT1 [21].Therefore, to further characterize IL-21 effects on CpG methylation of key EBV genomic promoters, we performed MeDIP-qPCR analysis on Mutu I or Kem I Burkitt cells treated with IL-21 for 1 or 2 days.Interestingly, IL-21 downmodulated the high level of DNA methylation at the LMP1 and C promoters, but not at the LMP2 promoter in either Mutu I or Kem I (Figs 3E and S5E).Additional epigenetic marks may maintain Cp and LMP2p silencing upon IL-21 stimulation in the latency I context, including those driven by STAT-containing repressive complexes.In support of a STAT3 role in modulation of LMP1p methylation downstream of IL-21, we did not observe diminished LMP1p methylation levels in STAT3 depleted Mutu I cells upon IL-21 treatment (Fig 3F).

Fig 3 .
Fig 3. STAT3 roles in GC cytokine mediated LMP1 de-repression in latency I B-cells.(A) Immunoblot analysis of WCL from latency I Mutu I or Kem I B cells pre-treated with DMSO or JAKi (200 ng/ml) for one hour, followed by treatment with the indicated cytokines for one day.(B) Immunoblot analysis of WCL from Mutu I expressing control sgRNA or sgRNA targeting the indicated STAT transcription factor gene, mock treated or treated with IL-4+CD40L for one day.(C) Immunoblot analysis of WCL from Mutu I expressing control sgRNA or sgRNA targeting the indicated STAT transcription factor gene, treated with IL-10 or IL-21 for one day.(D) Immunoblot analysis of WCL from Mutu I mock treated or treated with IL-21 for one or two days.(E) MeDIP q-PCR analysis of LMP1 promoter methylation in Mutu I (left) or Kem I (right) mock treated or treated with IL-21 for one or two days.Shown are mean ± SD values of % input from n = 3 replicates.(F) MeDIP-qPCR analysis of LMP1p in Mutu I with control or STAT3 targeting sgRNA, mock treated or IL-21 treated for one day.*p < 0.05; **p < 0.01; ***p < 0.001.Cytokines were used at 50 ng/ml.Blots are representative of n = 3 replicates.https://doi.org/10.1371/journal.ppat.1011939.g003 (Fig 4A), we performed chromatin immunoprecipitation (ChIP) and qPCR analyses in LCLs mock treated or treated with FDC-derived IL-15 or Tfh-derived IL-21.IL-21 increased STAT3 occupancy at the S3 site, located at approximately 600 base pairs (bp) upstream of LMP1p, and to a lesser extent at the S2 and S1 sites, located at approximately 500 and 100 bp upstream of LMP1p (Fig 4B).Interestingly, IL-15 instead downmodulated STAT3 occupancy at S2 and S3, consistent with the observation that it does not hyper-induce LMP1 in latency III.By contrast, IL-15 but not IL-21 significantly increased STAT5 occupancy at S1-S3 (Fig 4C).These results further support the hypothesis that IL-21 driven STAT3, and potentially STAT3 homodimers, are major drivers of LMP1 hyper-induction.To characterize STAT roles in LMP1 promoter histone epigenetic regulation, we next performed ChIP-qPCR analysis in control versus CRISPR-edited LCLs.In control LCLs, IL-15 and to a greater extent IL-21 increased LMP1p histone 3 lysine 27 acetylation (H3K27Ac), a mark which correlates with promoter activation.By contrast, IL-15 and IL-21 failed to upregulate LMPp H3K27Ac level in LCLs depleted for STAT3, STAT5A and STAT5B (Fig 4D).Since we recently found a role for the polycomb repressive complex (PRC1) I histone 2A lysine 119 ubiquitin (H2AK119Ub) mark in repression of LMP1 expression [21], we next examined GC cytokine and STAT roles on LMP1p H2AK119Ub levels in LCLs.IL-15 and to a greater extent IL-21 significantly diminished H2AK119Ub abundance in control, but not STAT3/5A/5B KO GM12878 (Fig 4E).Despite lack of appreciable LMP2A hyper-induction, IL-21 nonetheless increased H3K27Ac levels in GM12878 control and STAT3/5A/5B edited LCLs (Fig 4F).Interestingly, IL-21 hyper-induced H2AK119Ub repressive marks at LMP2p in both control and STAT3/5A/5B edited cells (Fig 4G).Given PRC1 roles in repression of LMP expression, this result suggests a potential mechanism by which LMP1 but not LMP2A is hyper-induced in IL-21 treated B-cells, and are consistent with a model in which STAT3/5 occupy LMP1 but not LMP2 promoter sites.We did not observe decreases in the repressive histone 3 lysine lysine 9 dimethyl (H3K9me2) or trimethyl (H3K9me3) marks with either IL-15 or IL-21 treatment in control or STAT KO LCLs (S6A and S6B Fig).However, repressive histone 3 lysine 27 trimethyl (H3K27me3) repressive marks increased somewhat upon IL-15 or IL-21 treatment in STAT3/5A/5B triple edited LCLs (S6C Fig).Similar effects were observed at the LMP2 promoter, though IL-21 increased the repressive H3K9me3 mark in both control and STAT3/5A/5B edited cells (S6D-SF Fig), potentially contributing to the lack of IL-21 driven LMP2A hyper-induction.We next characterized IL-21 epigenetic effects on the LMP1 promoter in latency I B-cells, given the observation that IL-21 strongly activates STAT3 phosphorylation and de-represses LMP1 expression, whereas other GC cytokine stimuli did so comparatively weakly.As anticipated, IL-21 significantly increased H3K27Ac at the LMP1 promoter in Mutu I cells (Fig 4H).Interestingly, STAT3 was necessary for this IL-21 driven epigenetic remodeling, as STAT3 depletion prevented IL-21 driven H3K27Ac activating mark at the LMP1 promoter (Fig 4H).Similarly, IL-21 significantly diminished the repressive H2AK119Ub and H3K9me2 marks at the LMP1 promoter in a STAT3 dependent manner (Figs 4I and S7A).By contrast, IL-21 did not significantly alter repressive H3K9me3 or H3K27me3 marks at the latency I LMP1 promoter (S7B Fig).Interestingly, IL-21 did not significantly alter activating or repressive histone marks at the Mutu I LMP2 promoter (S7C-S7F Fig).These results indicate that the absence of STAT3 signaling is important for silencing LMP1 expression in latency I, with relevance to the transition from latency II to latency I.

Fig 6 .
Fig 6.IL-15 and IL-21 remodeling of latency III gene expression in newly infected primary human B cells.(A) Immunoblot analysis of WCL from primary human B cells at the indicated days post infection (DPI) by the Akata EBV strain.(B) Immunoblot analysis of WCL from primary human B cells at 7 DPI, which were then mock treated or treated with the indicated cytokines for six days.(C) Immunoblot analysis of WCL from primary B cells that were treated with DMSO or JAKi (200 ng/ml) for two days at 4, 7 or 10 DPI.GM12878 WCL was included as a control.(D) Primary human B-cell transformation assay characterizing effects of DMSO vs JAKi (200 ng/ml) treatment on primary human B-cell outgrowth following infection by Akata EBV.Fitted non-linear regression curves are presented as mean ± SD from n = 3 replicates, *p < 0.05; **p < 0.01.Blots are representative of n = 3 replicates.Cytokines were used at 100 ng/ml.https://doi.org/10.1371/journal.ppat.1011939.g006

Fig 7 .
Fig 7. Model of EBV latency promoter epigenetic remodeling by GC cytokine driven JAK/STAT signaling.Cytokines secreted by germinal center FDC and Tfh cells trigger JAK/STAT signaling to drive STAT homodimer or heterodimer nuclear translocation.STAT dimers bind to the C promoter together with a co-repressor to increase levels of repressive 5-methylcytosine (5-mC), H3K9me2/3 and H2AK119Ub epigenetic marks, while decreasing activating H3K27Ac marks.C promoter inactivation decreases latency III EBNA expression.By contrast, STAT dimers bind to the LMP1 promoter together with a co-activator to drive epigenetic remodeling to increase levels of LMP1 promoter H3K27Ac and to decrease levels of H2AK119Ub, which supports LMP1 expression despite concurrently increased 5mC levels.By contrast, GC cytokines increase the abundances of LMP2 promoter repressive H2AK119Ub, H3K9me3 and 5mC marks to downmodulate LMP2 expression from levels observed in latency III.(BioRender was used to create the schematic models).